Insect pests and natural enemies associated with lettuce Lactuca sativa L. (Asteraceae) in an aquaponics system

Although food is produced in aquaponics systems worldwide, no information is available on the occurrence of insect pests and natural enemies in aquaponic lettuce, Lactuca sativa L. In this study, a survey was carried out in an aquaponic system combining lettuce with lambari, Astyanax altiparanae (Garutti & Briski), aiming to determine the insect pests and natural enemies associated with this system. We also determined the predominant insect species and the effect of meteorological factors on their populations. Insect abundance was estimated by visual sampling during 13 cultivation cycles, totaling 27 sampling dates. The meteorological factors considered were air temperature and relative humidity, and their effects were determined using the Pearson correlation. The thrips Frankliniella schultzei (Trybom) and Caliothrips phaseoli (Hood) and the aphid Aphis spiraecola (Patch) predominated. Ambient temperature and relative humidity were essential factors affecting C. phaseoli and F. schultzei. The natural enemies found on the lettuce plants were the thrips Franklinothrips vespiformis (Crawford) and Stomatothrips angustipennis (Hood) and the ladybugs Cycloneda sanguinea L., Eriopis connexa (Germar), and Hippodamia convergens (Guérin-Méneville). These results constitute the first step for a lettuce-integrated pest-management program in aquaponics systems.


Aquaponic system
The aquaponics system included the following components: four hydroponic beds, each with an area of 2 m 2 and continually recirculated with a submerged pump.The system had two circular polyethylene tanks (500 L): a fish tank and a sump containing the water pump that distributed water to the entire system (Fig. 1).
Each hydroponic bed had Cinexpan® expanded clay as a substrate, where 15-day-old lettuce seedlings cv.Vanda obtained from a commercial vegetable seedling production nursery were transplanted.The lettuce was harvested after four weeks, according to 3 .Lettuce was cultivated during 13 cycles, which were carried out continuously, from February to September 2021.During April, the system was undergoing maintenance and lettuce cultivation resumed in May.The aquaponics system was maintained weekly, including regular cleaning of the sedimentation filter, water replenishment, and periodic observation of disease and stress symptoms in the fish.
A total of 300 juvenile lambaris (A.altiparanae) were kept in a circular polyethylene tank (500 L), with water entering in a tangential flow that helped to remove the fish waste.The water-flow calibration ensured a circulation water-flow rate of 100% per hour were used.Their mean initial weight was 3.0 g (δ = 0.44 g, n = 30) and reached a mean of 36 g (δ = 6.5 g; n = 30) at the end of the experiment.The final estimated total fish biomass (273 individuals) was approximately 9.83 kg, with a final density of 12.3 kg of fish.m -3 .Fish were fed with extruded feed containing 40% crude protein (CP) and 1.8-2.0mm in diameter.The feed was offered once or twice a day ad libitum, five days a week.During the experimental period, a total of 12.5 kg of feed was consumed by the fish, reaching a feed efficiency of 70%.
We emphasize that the lambari fish were not the target of evaluations in the present study, i.e., these organisms were kept without being manipulated during the research.The Postgraduate Course Committee "Plant and Animal Health, Food and Environmental Safety in the Agribusiness" at the Biological Institute evaluated and approved the experimental protocols related to lambari fish and lettuce plants.All methods were performed in accordance with the relevant guidelines and regulations of the Biological Institute, São Paulo, SP, Brazil.The lambari fish farming followed the recommendations of the Brazilian Federal Law 11,794 (https:// www.plana lto.gov.br/ ccivil_ 03/_ ato20 07-2010/ 2008/ lei/ l11794.htm).This study followed Arrive guidelines (https:// arriv eguid elines.org).
During the experimental period, iron deficiency in the lettuce plants was monitored based on leaf color change, according to 23 .To prevent this, the commercial formulation Rexolin, containing iron chelated with EDDHA 6%, was applied in a concentration of 2 mg L -1 .Chelated iron was reapplied every 2 months.The ammonia, nitrite, and nitrate concentrations in water were quantified using the API Freshwater Master Test Kit, which uses colorimetric methods to determine the concentration of these chemical compounds.These values served predominantly as a reference point, ensuring that no toxic compounds such as ammonia or nitrite were present.The nitrate concentration was sufficient to promote plant growth.Limnological water parameters (pH, temperature, oxygen saturation, and electrical conductivity; Table 1) were monitored and recorded weekly, in the morning, using an Akso AK88 multiparameter probe.The water was maintained at pH = 7.0 by applying the bases calcium hydroxide and potassium hydroxide alternately at weekly intervals.The fresh weight of 382 plants from 10 harvest periods was determined.The mean fresh weight (including the shoot and roots of each plant) was 170 g (δ = 37.5 g), while the mean fresh weight of the aerial part of the plant was 151 g (δ = 36.3g).

Statistical analyses
The faunistic coefficients of dominance, frequency, constancy, and abundance of the insects were determined by faunistic analysis, using the ANAFAU software 24 The species were classified as predominant when they reached the highest values of the faunistic coefficients, following Silveira 25 .
The effects of the meteorological factors (maximum, minimum, and mean temperatures, maximum and minimum relative humidities, and rainfall) on the abundance of predominant species was examined by the Pearson correlation, using the IBM SPSS Statistics 20 program 26 .The analysis was based on the total number of individuals of the insect species found on each sampling date.Regarding abiotic factors, we used the mean temperature and relative humidity recorded seven days before the sampling date, and the total rainfall in that period.Python IDE (integrated development environment) Jupyter Lab (version 3.7.4for Windows) software was used to draw the scatter diagrams representing the correlation between thrips occurrence and meteorological factors.The meteorological data were obtained from the Agência Brasileira de Meteorologia Ltda.(Climatempo Agency).
The population fluctuations of the predominant species of insect pests were obtained from graphs relating the number of individuals to mean temperature, relative humidity (maximum and minimum), and rainfall.

Statement of compliance
The authors declare that the experimental studies involving the collection and use of lettuce plants in this research are in accordance with relevant institutional, national, and international guidelines and regulations.

Abundance of insect pests
A total of 4078 individual phytophagous insects belonging to the aphids and thrips groups were captured, during 13 growing cycles of the lettuce aquaponics (Table 2).

Population fluctuations and effects of meteorological factors
Except for June and July, A. spiraecola occurred in most of the samples taken from February to September (Fig. 2).During June and July, the mean ambient temperature was 19.4 °C, relative humidity 54.9%, and total rainfall 64.0 mm.In the remaining months these factors reached 24.1 °C, 57.6%, and 188.0 mm, respectively.The correlation between A. spiraecola and the meteorological factors was not significant (Table 3).
The population fluctuations of C. phaseoli and F. schultzei were similar throughout the study period.These thrips were usually abundant from February to May, June to July, and September, coinciding with a period of low rainfall (Fig. 3).The highest population peak of C. phaseoli and F. schultzei occurred, respectively, in July and September.

Ocurrence of natural enemies
Six species of predatory insects representing two orders were captured and identified as follows:

Discussion
Aphids are economically important pests of lettuce.Besides the damage and the loss of lettuce quality from their feeding, aphids are vectors of viruses that cause lettuce diseases 29,30 .Although in this study, five species of aphids were found associated with lettuce, only the green citrus aphid, A. spiraecola, stood out as very abundant and frequent.However, this species was not found in June and July, the austral winter.Aphis spiraecola is one of the most abundant species in lettuce crops 31 but has not been reported to damage lettuce.Aphis spiraecola is polyphagous, occurs worldwide, and is a recognized pest of citrus, apple trees, and ornamental plants, and a vector of several species of phytoviruses 32,33 .The green peach aphid, M. persicae, is an important pest worldwide.This aphid causes up to 50% loss of yield in vegetable crops, due to phytovirus transmission 34 .In Brazil, M. persicae comprised 38% of individuals of three aphid species found in hydroponic lettuce 35 .The potato aphid, M. euphorbiae, has a wide host range and is also an important pest worldwide, due to its transmission of several phytoviruses 36 .In Brazil, M. euphorbiae is one of the main pests of lettuce in protected cultivation, and a vector of the LMV virus 37 .This aphid has been reported in hydroponic lettuce in São Paulo state 35 and in lettuce fields in Paraná state 34 .Additionally, H. lactucae and P. bursarius are common in many countries [38][39][40] and can be vectors of plant viruses in lettuce [41][42][43] .
In the present study, the small number of aphids found on the lettuce plants may be related probably to the lettuce stiffness leaf.This lettuce cultivar in aquaponic system showed leaves with a stiffer texture than when it was grown in soil.Ibrahim and Zuki 44 found that the Grand Rapid lettuce cultivar had a significantly higher percentage of fiber in an aquaponic system compared to lettuce grown in hydroponics and soil.According to 45 the dietary fiber found in vegetables, cereals, and fruits is composed of polysaccharides, cellulose, hemicellulose, lignin, pectins, gums, and oligosaccharides, which are recognized as resistance factors of plants to insect pests in cotton, potatoes, and forage plants 46-48; lignin is a particular component of resistance to aphids.
Studies on varietal resistance to pests are essential to the genetic improvement of lettuce 29 .However, the possible differences in resistance to lettuce pests in aquaponic, hydroponic, and soil systems remain to be clarified.We suggest investigating the relationship of dietary-fiber components of lettuce cultivars to the resistance of this vegetable against aphids and thrips, with a focus on developing strategies to manage pests in aquaponic systems.
Although the diversity of pests and natural enemies in lettuce grown in field and hydroponic conditions has been widely studied 34,35,[49][50][51][52] , we are unaware of published studies on the occurrence of these organisms in aquaponic lettuce.This is an essential step in the development of an innovative system such as aquaponic lettuce.
In the present study, F. schultzei and C. phaseoli constituted 99% of the captured specimens among the five thrips species found in lettuce.Frankliniella schultzei and C. phaseoli are considered the most abundant and harmful in lettuce grown in the field and hydroponically 34,52 .The former is a vector of tospoviruses (genus Tospovirus, family Bunyaviridae), an important group of viruses affecting many plant species, including lettuce.Furthermore, when feeding on lettuce leaves, F. schultzei causes chlorotic spots that reduce the commercial quality 34,42,53,54 .The low incidence of Gynaikothrips sp., F. insularis, H. gowdeyi, E. mexicanus, and Pseudophilothrips sp. may indicate that these thrips occurred incidentally in the lettuce plants.Only F. insularis has been reported to damage lettuce.Frankliniella insularis has a wide distribution in the Americas and is reported to cause considerable damage to lettuce on the islands of Guadeloupe and Martinique, and also as a secondary pest of citrus in Brazil 55,56 .Haplothrips gowdeyi is widely distributed in tropical and subtropical countries, occurring in flowers of a wide variety of plants 56 .Echinothrips mexicanus occurs in lettuce, and cassava 52,57 .No published studies have reported Pseudophilothrips sp. on lettuce.However, the genus Pseudophilothrips is a pest of guava, and its damage compromises the fruits' aesthetic value and increases the plant's susceptibility to fungal infestation 58 .The genus Gynaikothrips includes 16 species associated with Ficus (Moraceae) that cause considerable damage to plant leaves 59 .
Environmental temperature was the most important abiotic factor affecting the incidence of C. phaseoli in lettuce, while temperature and relative humidity were essential factors affecting F. schultzei.According to 60 , temperature and rainfall are meteorological factors responsible for up to 65% of the numerical variation of thrips in an agroecosystem.In the present study, the abundance of C. phaseoli decreased in periods with mean temperatures above 30 °C during summer and late winter, while its maximum population peak occurred in winter, with a mean temperature of 26.6 °C.The two highest population peaks of F. schultzei coincided with rising The lowest population densities of C. phaseoli and F. schultzei occurred in periods of more intense rainfall.Rainfall contributes significantly to the mortality of insect pests, affecting them both directly (washing and drowning individuals) and indirectly by favoring the occurrence of entomopathogenic fungi [61][62][63][64] .The aquaponic system used here was in a semi-field environment where the lettuce plants were not directly exposed to rain.As mentioned above, meteorological factors such as temperature, and especially rainfall, can have a significant impact on thrips abundance 60,[65][66][67] .The significant negative correlation between relative humidity and F. schultzei obtained here may indicate an indirect influence of rain that limited the flight capacity of thrips, reducing dispersal and, consequently, colonization of lettuce plants in periods of rainfall.
Barrière et al. 29 highlighted the importance of favorable climatic conditions for the incidence of pests in lettuce crops.In view of the present results, it is essential to conduct studies on the influence of meteorological factors such as temperature, relative humidity, and rainfall on C. phaseoli and F. schultzei.Understanding the effects of abiotic factors on a thrips population is necessary for an effective and sustainable control strategy, aiming to minimize thrips damage and increase lettuce quality and productivity.Among the species of predatory thrips collected on lettuce, F. vespiformis feeds on small arthropods, including mites, whiteflies, and thrips.The species is an important control agent for thrips of the genus Scirtothrips that attack avocado trees 56 .Stomatothrips angustipennis occurs only in Brazil, but there is no information about its economic importance in agriculture.However, the species is a predator of mites and other thrips species 56 .
The low density of ladybugs found here may be related to the low occurrence of aphids on the plants, since aphids are essential food for most species of ladybugs 76 .The lack of vegetation near the aquaponic system may also have contributed to the low incidence of these natural enemies.A plant shortage around aquaponic lettuce can be modified by providing sources of food and shelter to maintain and increase populations of natural enemies 77 .According to 78 , diversification of plant species can increase the abundance, diversity, and dispersal of alternative prey, enhancing generalist predator populations.Sengonca et al. 79  This study indicated that thrips and aphids are significant pests of aquaponic lettuce, and natural enemies were represented by ladybugs and predatory thrips, occurring in low density.On the other hand, we are aware of no published studies on control of pests in aquaponic lettuce 6 , and conservation biological control is appropriate for aquaponics technology.This situation opens avenues of opportunity for developing pest-management strategies such as habitat manipulation, which has wide applications in agriculture 80 .For example, to increase ladybug populations in aquaponic lettuce, we can recommend establishment of other plant species that do not host lettuce pests and have periods of intense flowering in the vicinity of the system.
reported that plants of Artemisia vulgaris L. (Asteraceae), Tanacetum vulgare L. (Asteraceae), and Urtica dioica L. (Urticaceae) adjacent to a lettuce crop increased the number of larvae and adults of Coccinella septempunctata L., Adalia bipunctata L., and Propylea quatuordecimpunctata L., which reduced the populations of M. persicae, Nasonovia ribisnigri Mosley, and M. euphorbiae.

Sampling insect pests and natural enemies The
abundance of insect pests and natural enemies was determined by visual sampling of the lettuce plants during 13 cultivation cycles, from February to September 2021.During the sampling period, 10 plants per hydroponic bed were randomly selected each week.Insect pests and natural enemies were captured with an entomological aspirator and glass tube (height 8.0 cm, diameter 2.5 cm), respectively.The insect pests and natural enemies were transferred to the Laboratory of Entomology and Biological Control (LECB), Ribeirão Preto, São Paulo, Brazil.Aphid and thrips specimens were separated using a number zero camel-hair brush and transferred to Eppendorf tubes containing 70% ethanol.The thrips species were identified by Élison Fabrício B. Lima, Laboratory of

Table 2 .
Faunistic coefficients of aphid and thrips species on lettuce plants in an quaponics system, Cravinhos, São Paulo state, Brazil.

of insects captured Sampling rate Dominance Frequency Constancy Abundance
was predominant, found on 9 of the 27 sampling dates, totaling 52% of all aphids collected.The other aphid species were non-dominant, frequent, or infrequent.